維生素K3的激發三重態與色氨酸、酪氨酸電子轉移氧化反應的激光閃光光解研究

唐睿智 李海霞 劉艷成 張 鵬 曹西艷 王文鋒

(1中國科學院上海應用物理研究所,上海201800; 2中國科學院研究生院,北京100049; 3湖南科技大學化學化工學院,湖南湘潭411201)

維生素K3的激發三重態與色氨酸、酪氨酸電子轉移氧化反應的激光閃光光解研究

唐睿智1,2李海霞1,2劉艷成1,2張 鵬1,2曹西艷1,3王文鋒1,*

(1中國科學院上海應用物理研究所,上海201800;2中國科學院研究生院,北京100049;3湖南科技大學化學化工學院,湖南湘潭411201)

利用時間分辨的激光閃光光解技術研究了乙腈-水混合溶液(1:1,V/V)中2-甲基萘醌(通常稱為維生素K3)的激發三重態對色氨酸、酪氨酸的光敏氧化機理.通過瞬態吸收光譜的變化可以推斷維生素K3的激發三重態可以與色氨酸、酪氨酸發生電子轉移反應,反應形成的維生素K3陰離子自由基的吸收峰可以直接從瞬態吸收譜圖中觀察到.維生素K3與色氨酸、酪氨酸的電子轉移反應的速率分別為1.1×109和0.6×109L·mol-1·s-1.吉布斯自由能(ΔG)的計算結果表明維生素K3的激發三重態與色氨酸、酪氨酸電子轉移反應在熱力學上是可行的.

維生素K3;色氨酸;酪氨酸;激光閃光光解

1 Introduction

The photosensitized oxidation of biomolecule by photo-oxidation reaction has received increased attention in recent years. The free radical theory of aging1,2shows that aging is a result of destructive effects on biomolecule.Proteins are major potential targets for oxidation because they comprise approximately 68%of the dry mass of cells and tissues.Aromatic amino acid residues in proteins,especially tryptophan(TrpH)and tyrosine (TyrOH),are easily oxidized.3,4Thus,the reactions of TrpH and TyrOH with photosensitizers have received considerable atten-tion in the field of proteins photo-oxidation.5-7Oxidative damage to proteins occurs via complex chemical reactions that lead to changesin the proteins?structure and the rupture of the polypeptide chain as well as protein cross linking.8The oxidative damage of proteins plays a critical role in the development of Alzheimer?s disease,Parkinson?s disease,cancer,and aging.9-12

Vitamin K3,commonly known as 2-methyl-1,4-naphthoquinone(MQ),is one of the widely used endogenous photosensitizers with antihemorrhagic activity.13MQ is present in vegetables and plays an active role in the photosynthetic mechanism. It also participates in cellular respiration as an electron transporter,and in oxidative phosphorylation14-17as an effective electron carrier and electron transfer agent.18,19Many studies have reported on the mechanisms of electron transfer oxidation of nucleotides by the triplet state of MQ(3MQ*)via laser flash photolysis technique.20-23Charge transfer reactions between (3MQ*)and polyguanylic acid(polyG),guanine nucleotide (dGMP)demonstrated that polyG radical cation,dGMP radical cation,and MQ radical anion can be detected simultaneously. In recent years,Basu et al.24-26has been concerning and studying magnetic field effects(MFEs)and medium-dependent effect on the photochemistry reactions between MQ and DNA, RNA bases,the results indicated that H atom transfer and electron transfer are the operative mechanisms depending upon the medium.

However,to our knowledge,compared with the research on the triplet state of MQ reacting with nucleosides,fewer studies have discussed the oxidation of proteins by the triplet state of MQ,owing to its complicated structure and mechanism.Therefore,it is a significant task to elucidate the mechanisms of interaction between the triplet state of MQ and protein monomer units or complexes.In the current study,TrpH and TyrOH are selected as models to observe the photo-oxidation mechanisms.We attempted to investigate the photo-processes of MQ with TrpH and TyrOH in acetonitrile/water(1:1,V/V)solutions using the 355 nm laser flash photolysis.The possible mechanisms are proposed and the related rate constants are obtained in this study.

2 Experimental

2.1 Materials

Vitamin K3(MQ),tryptophan(TrpH),tyrosine(TyrOH),and acetonitrile were purchased from Sigma-Aldrich.All the chemicals were analytically pure and used as received without further purification.All solutions were prepared freshly with pure water provided by Millipore purification system before each experiment.Samples were bubbled with high-purity N2or O2(99.999%)for 20 min before laser flash photolysis.All solutions were buffered with phosphate(1×10-3mol·L-1,pH 7.0).

2.2 Methods

Nd:YAG laser(NL303HT,EKSPLA,Lithuania)provided 355 nm pulse with duration of 5 ns and the maximum energy of 8 mJ per pulse was used as the pump light source.A xenon lamp was employed as detecting light source.The laser beam and analyzing light passed perpendicularly through a quartz cell.The transmitted light entered a monochromator equipped with an R955 photomultiplier.The output signal from the HP54510B digital oscillograph was transferred to a personal computer for study.The laser flash photolysis setup has been previously described.27

3 Results and discussion

3.1 Photoreaction of MQ with TrpH in acetonitrile/

water(1:1)solution

As shown in Fig.1,in the transient absorption spectrum after 355 nm laser flash photolysis of the N2-saturated acetonitrile/ water(1:1)solution containing 0.1×10-3mol·L-1MQ,a maximum absorption band at 370 nm was observed.The triplet state absorption of MQ(at 370 nm)decays by a first-order process with a rate of 3.4×105s-1in the N2-saturated acetonitrile/ water(1:1)solution containing 0.1×10-3mol·L-1MQ.The maximum transient absorption at 370 nm can be quenched efficiently by O2(Fig.1 inset).This result was similar to the reported triplet state absorption spectrum of MQ.24,28

Fig.2 shows the transient absorption spectra after 355 nm laser flash photolysis of the N2-saturated acetonitrile/water(1:1) solution containing 0.1×10-3mol·L-1MQ and 0.2×10-3mol· L-1TrpH.Under such conditions,the transient absorption of the triplet state of MQ appeared immediately at the end of the laser flash pulse,and decayed significantly faster in the presence of TrpH.Following the decay of the triplet state of MQ at 370 nm,a spectrum with maximum absorption bands at 390 and 510 nm appeared.The energy of the triplet(ET)of MQ is about 239 kJ·mol-1,29while the ETof TrpH is 297 kJ·mol-1.30Therefore,the formation of the triplet state of TrpH via energy transfer from the triplet state of MQ is unlikely to occur.According to previous reports,the maximum absorption of the MQ anion radical22and Trp?31,32were located at 390 and 510 nm,respectively.Consequently,the transient absorption spec-tra resulting from MQ reacting with TrpH can be explained by electron transfer from TrpH to the triplet state of MQ,generating MQ anion radical and TrpH cation radical(TrpH+?).According to the pKagiven in the literature,7the TrpH+?(pKa=4.3) turned into a neutral radical Trp?by deprotonation under the experimental conditions.As shown in Fig.2(inset),the formation of MQ anion radical and Trp?radical was synchronous with the decay of the triplet state of MQ,which further demonstrated the electron transfer reaction between the triplet state of MQ and TrpH.

Fig.1 Transient absorption spectra recorded at(▲)0.1 μs and (●)1 μs after 355 nm laser photolysis of 0.1×10-3mol·L-1MQ in N2-saturated acetonitrile/water(1:1)solutioninset:transient time trace recorded at 370 nm in N2-saturated(a), O2-saturated(b)acetonitrile/water(1:1)solution

Fig.2 Transient absorption spectra recorded at(▲)0.1 μs and (●)2 μs after 355 nm laser photolysis of N2-saturated acetonitrile/ water(1:1)solution containing 1×10-3mol·L-1MQ and 2×10-3mol·L-1TrpHinset:transient kinetic traces recorded at(a)390 nm and(b)510 nm

Varying the TrpH concentration(0.2×10-3-0.7×10-3mol· L-1),the decay of3MQ*was accelerated in the presence of TrpH,with rates proportional to the concentrations of TrpH (Fig.3),the rate constant of the reaction was determined to be 1.1×109L·mol-1·s-1by monitoring the formation of MQ anion radical(Table 1).Thus,the mechanism of TrpH with the triplet state of MQ can be illustrated as the following:3MQ*+TrpH→MQ-●/MQH●+TrpH+●/Trp●

3.2 Photoreaction of MQ with TyrOH in acetonitrile/ water(1:1)solution

Fig.3 Dependence of kobsat 390 nm on the concentration of TrpH

Table 1 Rate constants and free energy changes(ΔG)of electron transfer from TrpH and TyrOH to the triplet state of MQ

Fig.4 shows the transient absorption spectra after 355 nm laser flash photolysis of the N2-saturated acetonitrile/water(1:1) solution containing 0.1×10-3mol·L-1MQ and 0.2×10-3mol· L-1TyrOH.Similar to the photoreaction of MQ with TrpH,the transient absorption of the triplet state of MQ appeared immediately after laser flash pulse and decayed significantly faster in the presence of TyrOH.However,after the decay of the triplet state of MQ,a spectrum with only a maximum absorption at around 390 nm formed.The transient absorbance of TyrOH+?/TyrO?33(at 410 nm)could not be observed clearly due to overlapping with that of the MQ anion radical or its lower absorption coefficient.The formation of the MQ anion radical was also observed to be synchronous with the decay of the triplet state of MQ(Fig.4 inset).The formation of triplet state ofTyrOH via energy transfer from triplet state of MQ is also unlikely to occur because the ETof TyrOH(342 kJ·mol-1)30is higher than that of MQ(239 kJ·mol-1).Therefore,It is concluded that the electron transfer from TyrOH to the triplet state of MQ also occurs.The rate constant of MQ with TyrOH was acquired in a manner similar to the photoreaction of MQ with TyrOH(Fig.5),the rate constant of the reaction was determined to be 0.6×109L·mol-1·s-1.The reaction mechanism of TyrOH with the triplet state of MQ can be illustrated as following:

3.3 Calculation of the free energy changes(ΔG)for the electron transfer reactions

Fig.4 Transient absorption spectra recorded at(▲)0.1 μs and (●)3 μs after 355 nm laser photolysis of N2-saturated acetonitrile/water(1:1)solution containing 0.1×10-3mol·L-1MQ and 0.2×10-3mol·L-1TyrOHinset:transient kinetic trace recorded at 390 nm

To further prove the thermodynamic feasibility of the reaction,the free energy changes(ΔG)of the electron transfer between the triplet state of MQ and TrpH or TyrOH can be calculated by the Rehm-Weller equation:34where Eox(in V)and Ered(in V)are the oxidation potentials of the donor and the reduction potential of the acceptor,respec-tively,and E0,0(in kJ·mol-1)is the triplet state energy of MQ. Here,e2/εd is the coulombic term,equal to 0.024 eV in acetonitrile/water(1:1)solution.35The Eredand E0,0values of MQ are-0.77 V(vs saturated calomel electrode(SCE))and 239.3 kJ·mol-1,respectively.29The Eoxvalues of TrpH and TyrOH are 1.01 and 0.93 V(vs normal hydrogen electrode(NHE)),respectively.36Therefore,the ΔG values obtained for TrpH and TyrOH are-69.6 and-79.2 kJ·mol-1,respectively(Table 1). Thus,the photo-induced electron transfer from TrpH and TyrOH to the triplet state of MQ is thermodynamically favorable.

Fig.5 Dependence of kobsat 390 nm on the concentration of TyrOH

4 Conclusions

The above experimental results indicated that the triplet state of MQ reacted with TrpH and TyrOH by electron transfer with the formation of MQ anion radical or oxidized radicals of Trp?and TyrO?.The rate constants were determined to be 1.1× 109and 0.6×109L·mol-1·s-1for TrpH and TyrOH,respectively, and the free energy changes show that the electron transfer reactions are thermodynamically feasible in our experiment. Knowledge of the electron transfer oxidation of TrpH and TyrOH by the triplet state of MQ is important to better understand structural effects on the photochemical behavior of proteins with the triplet state of MQ.

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Laser Flash Photolysis Study on Electron Transfer Oxidation Reaction of Tryptophan or Tyrosine with Triplet State Vitamin K3

TANG Rui-Zhi1,2LI Hai-Xia1,2LIU Yan-Cheng1,2ZHANG Peng1,2CAO Xi-Yan1,3WANG Wen-Feng1,＊
(1Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai 201800,P.R.China;2Graduate University of Chinese Academy of Sciences,Beijing 100049,P.R.China;3School of Chemistry and Chemical Engineering,Hunan University of Science and Technology,Xiangtan 411201,Hunan Province,P.R.China)

Laser flash photolysis was used to study the photosensitized oxidation mechanism of vitamin K3,commonly known as 2-methyl-1,4-naphthoquinone(MQ),with tryptophan(TrpH)or tyrosine(TyrOH)in acetonitrile/water(1:1,V/V)solution.The triplet state of MQ reacted with TrpH or TyrOH by electron transfer with the formation of a MQ anion radical,which was directly observed in the transient absorption spectrum.The rate constants of the electron transfer reactions were determined to be 1.1×109and 0.6×109L·mol-1·s-1for TrpH and TyrOH,respectively.The free energy changes(ΔG)of the reactions showed that the proposed electron transfer steps are thermodynamically feasible.

Vitamin K3;Tryptophan;Tyrosine;Laser flash photolysis

10.3866/PKU.WHXB201228213

*Corresponding author.Email:wangwenfeng@sinap.ac.cn;Tel:+86-21-39194602.

The project was supported by the National Natural Science Foundation of China(21173252).

國家自然科學基金(21173252)資助項目

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